The oxidative coupling reaction between phenol and 4-aminophenazone, also known as 4-aminoantipyrine, to give a red quinoneimine dye has been known for a long time, having been described by Emerson, J. Org. Chem. 8:417 (1943).
The reaction has gained popularity in clinical chemistry since the application made by Trinder, [Ann. Clin. Biochem, 6:24 (1969)] to the enzymatic determination of glucose, based on the reaction scheme: ##STR1## The chromogenic system phenol (including substituted phenols) +4-aminophenazone+peroxidase, referred to as the Emerson-Trinder system, is now used in the quantitative determination not only of glucose, but also of cholesterol and uric acid in serum, plasma or other biological fluids. Use of this system for the determination of glucose is disclosed in Meiattini, U.S. Pat. No. 3,886,045, now reissued as U.S. Pat. No. Re 29,498.
The general scheme of the reaction is the following: ##STR2##
Many phenols can be used in the Emerson-Trinder reaction. Examples of those most commonly used in clinical chemistry are phenol; p-hydroxybenzoate; 2,4-dichlorophenol; 3,5-dichloro-2-hydroxybenzenesulfonic acid. Likewise various substituted and unsubstituted napthols can be used.
The sample constituents which can be determined include glucose, cholesterol, uric acid or other metabolites which can be oxidized by a specific oxidase with contemporaneous formation of hydrogen peroxide. The oxidase is glucose oxidase for determining glucose; cholesterol oxidase for determining cholesterol (cholesterol ester hydrolase is also added to hydrolyze esterified cholesterol); and uricase for uric acid determinations.
The amount of dye formed is proportional to the concentration of the hydrogen peroxide and, therefore, to the concentration of the constituent in the sample. Thus, the concentration of the constituent in the sample can be obtained by a simple measurement of the absorbance of the reacted solution and comparison of such measurement to that of a known standard solution of the constituent.
The dye formed can be measured in the visible range, generally between 500 and 550 nanometers (nm) (depending on the phenol used); requiring only a colorimeter or a visible color range photometer.
The Emerson-Trinder chromogenic system suffers the major disadvantage that the oxidative coupling reaction is affected by reducing compounds and bilirubin, a metabolite which is usually present in serum in concentrations not higher than 1 milligram per deciliter (mg/dl), but which can reach very high levels (20 or more mg/dl) in some diseases. Levels of bilirubin higher than normal affect the enzymatic glucose, cholesterol and uric acid tests by decreasing the color of the reaction. Interference increases with the increase of the bilirubin level.
The explanation of negative interference of reducing compounds (e.g. ascorbic acid) is quite obvious, since they act chiefly as competitors with the chromogen in the peroxidase catalyzed reaction with hydrogen peroxide, or as bleaching agents on the color formed. The interference of reducing substances, however, is not a real problem, at least in serum, where ascorbic acid rarely exceeds 3 mg/dl.
In contrast, the interference by bilirubin is a signficant problem for the determination of metabolites in serum through the Emerson-Trinder chromogenic system, and represents a major negative aspect of this system in routine laboratory practice, where hyperbilirubinemic samples are frequently found.
The mechanism of reaction of bilirubin is quite complex and, as yet, not fully understood. The best approach so far afforded to the problem is that of Witte [Clin. Chem. 24:1778 (1978)], who ascribes the interference of bilirubin to one or more of the following factors: simple spectral effects, acting as an alternative peroxidase substrate, or destruction of peroxidase reaction intermediates.